NANOBIOTECHNOLOGYAn Interdisciplinary Challenge

Uwe B. SLEYTRCenter for NanoBiotechnology

University of Natural Resources and Applied Life Sciences Vienna

Center for NanoBiotechnologyUniversity of Natural Resources and Applied Life Sciences Vienna

Head: Uwe B. SleytrDevelopment of a supramolecular construction kit based on monomolecular crystalline protein lattices (S-layers) as patterning elements for life and non-life science applications.

www.biotec.boku.ac.at/znb.html

… exploitation of the results is performed in close collaboration with Nano-S

Research groups:

• Nanoengineering (Margit Sára)

• Nanoglycobiology (Paul Messner)

• Nanostructures (Dietmar Pum)

Definition of NanoBiotechnology

• Nanobiotechnology involves the processing, fabrication and packag-ing of organic or biomaterial devices or assemblies in which the dimen-sion of at least one functional comp-onent lies between 1 and 100nm.

• Nanobiotechnology is characterized by its highly inter-disciplinary nature and features a close collaboration between life-scientists, physical scientists, and engineers. courtesy of FEI-Company, NL

Converging Technologies

NT is not a „rebranding“ of older science !Its influence is revolutionary rather than evolutionary.

mod. after Pielartzik, Bayer

Nanosciences and Nanotechnology

„Self-assembly“

Microlithography

Which are the Basic Building Blocks in a Biomolecular Construction Kit?

Biological molecules (e.g. proteins, lipids, glycans, nucleic acids)

Chemically or genetically modified molecules

Chemically synthesized molecules

Key Capabilities

Supramolecular design

Self-assembly strategies and morphogenesis (sequential assembly routes)

Patterning elements

• Monomolecular arrays• Vesicles• Tubes

Combination of "top-down“ and "bottom-up“ strategies

Functionalization of supramolecular structures

Characterization

Basic Structures (Patterning Elements) for Generating Complex Supramolecular Structures

• DNA

• Monomolecular crystalline bacterial cell surface layers (S-layers)

Nanoscale Assembly and Manipulation ofBranched DNA (Ned Seeman, NY University)

http://seemanlab4.chem.nyu.edu/homepage.html

Bacterial Surface Layer Proteins (S-layers)

AFM image of an S-layer with square (p4) lattice symmetry (d=13.1nm) reassembled on a silicon wafer.

Center for NanoBiotechnology and BOKU, Vienna, Austria

S-layers are crystalline, monomolecular (glyco)protein arrays representing one of the most commonly observed surface structures in eubacteria and archaea.

TEM of freeze etched preparations of bacterial cells with an S-layer showing square (left) and hexagonal (right) lattice symmetry.

Description of S-layers

For review see: Sleytr et al., 1999, Angew. Chemie Int. Ed., 38:1034-1054

S-layer Lattice Typesoblique square

hexagonal

p3 p6

p1 p2 p4

Center-to-center spacing of the morphological units in the range of 3 – 30 nm.

a 10 nm b 10 nmc 10 nm

Ultrastructure of S-layer Protein Lattices

Three dimensional image reconstruction of an S-layer with square (p4) lattice symmetry

AFM image of an S-layerwith square (p4) latticesymmetry

AFM image of an S-layerwith oblique (p2) latticesymmetry

Properties of S-layer Lattices

Patterning elements for a molecular construction kit

S-layer lattices are composedof identical species of subunits.

Pores passing through showidentical size and morphology.

Functional groups are alignedin well defined positions and show defined orientations.

Nano(bio)technological Applications of S-layers

• Isoporosity

• Sterically defined functionality

• Coating of Surfaces

• Components for supramolecular structures (molecular LEGO)

S-layer Ultrafiltration MembranesExploiting Isoporosity

S-layer

Microfiltration membraneas support

S-layer Ultrafiltration Membranes

Myoglobin (Mr 17 000)Carbonic anhydrase (Mr 30 000)Ovalbumin (Mr 43 000)Bovine serum albumin (Mr 67 000)

Sharp Cut Off

Conventional Functionalizationof Surfaces

Functionalization of S-layer Lattices(Binding of Molecules and Nanoparticles on S-layers)

Repetitive physicochemical properties

Pores in S-layers have identical sizeand morphology

Functional domains (native or genetically introduced) are repeated with the periodicity of the S-layer lattice leading to regular arrays of bound molecules and particles.

• Chemical methods• Design and expression of recombinant S-layer fusion proteins

Examples:

a b c100nm 100nm 200nm

Exact Orientation of Molecules and Functions

Native S-layer protein

S-layer fusion protein

Chemical or genetical functionalization

S-layer + Function (Antibody,

Streptavidin

10 nm

10 nm.

Enzyme , Antigen, Ligand)

Applications of S-layer Fusion ProteinsDiagnostic systems and label free detections systems (SPR, SAW, QCM-D)

High density affinity coatings (e.g. biocatalysis, blood purification (MDS))

Immunogenic and immunomodulating structures (e.g. anti-allergic vaccines)

Stabilization of functional lipid membranes

Functionalization of liposomes and emulsomes as targeting and delivery systems

Biomineralization

Binding of nanoparticles (e.g. molecular electronics, non linear optics)

S-layer Fusion Proteins

Silver binding peptideCobalt binding peptide

12 aa12 aa

rSbpA31-1068 / AG4 and AGP35rSbpA31-1068 / CO2P2

Heavy chain camel antibody117 aarSbpA31-1068 / cAb

Green fluorescent protein238 aarSbpA31-1068 / GFP

IgG-Binding domain116 aarSbpA31-1068 / ZZ

Affinity tag for streptavidin9 aarSbpA31-1068 / Strep-tag

Major birch pollen allergen116 aarSbpA31-1068 / Bet v1

Biotin binding118 aarSbsB1-889 / core streptavidinrSbpA31-1068 / core streptavidin

FunctionalityLength of funct.

S-layer fusion protein(selected from various constructs)

Mature proteins:Bacillus sphaericus CCM2177 variant A (SbpA): 1238 aaGeobacillus stearothermophilus PV72/p2 (SbsB): 889 aa

IgG

Microbead with bound SCWP

ZZ-domains

rSbpA31-1068

Primary circuit (blood)

Secondary circuit(plasma + microspheres)

Microspheres Based Detoxification-System

Cooperation with D. Falkenhagen, V. Weber et al.

Biomimetic Cell Membranes

TEM of an archaeal cell

http://sun.menloschool.org/~cw

eaver

Cell membrane: fluid mosaic model

Specific membrane functions

S-layer

Plasma-membrane1.selectivity 2.binding 3.opening / closing

Archaeal cell envelope structure

A supramolecular structure opti-mized in ~ 3,5 billions of years under extreme environmental conditions (120°C, pH 0, concen-trated salt solutions, 1100 bar).

S-layer Stabilized Solid Supported Lipid Membranes

S-layer

Functionalizedlipid membrane

S-layer

Solid support(e.g. Si-wafer, gold)

Schuster et al., 1998, Biochim. Biophys. Acta Biomembr.1370:280-288.Schuster et al., 2001 Langmuir 17: 499-503Schuster et al., 2002, Bioelectrochem. 55:5-7Schuster et al., 2003, Langmuir 19:2392-2397

S-layer proteins as:• stabilizing structures• tethering structures• ionic reservoir

Application Potential ofS-layer Supported Lipid Membranes

• Exploiting functional lipid membranes at meso- and macroscopic scale:

~30 % of all proteins found in various organisms are membrane proteins> 50 % of the proteins interact with membranes~ 15 % of the most sold drugs act on ion channels ~ 60 % of the ethical drugs affect membrane proteins

• Linking silicon technology and solid state physics with biological systems (e.g. coupling cells to surfaces)

Biosensors, HTScreening, Diagnostics, Lab-on-a-chip

bound functional molecules

S-layer protein

S-layerfusion

proteins transmembranefunction

Biomimetic VirusesS-layer Liposomes and Emulsomes

100nm

Potential Applications

• Artificial viruses (inclusion of nucleic acid) for gene therapy.

• Drug-targeting and drug-delivery.

• Vaccines and immune therapy.

• Transport of hydrophobic substances.

EM-Photograph of an S-layer coatedliposome.

HIV VirusSource: WellcomePhoto Library, Medical Art Service, München

Electron micrograph of ultrathin section demon-strating the internalization of S-layer coated lipid / plasmid particles into HeLa cells.

Photograph of HeLa cells expressing green fluor-escent protein after transfection with S-layer coated lipid/ pEGFP-C1 particles

S-layer Coated Lipid / Plasmid Particels

Cell patterningCellular lithography

Backgrounds of cell repulsive material

Grid patterns of cell friendly material

M. Scholl, et al, J. Neurosci. Meth. (2000) 104, 65-75

Summary

• Transdisciplinary strategies and integration of methods of Life- and Non Life Sciences as part of Converging Technologies.

• Optimal size (teams, equipment, infrastructure....). The BOKU has set priorities.

• Long-term development strategies (national and international grants).

• Adapted tertiary education: PhD-program involving different universities (Converging Universities).

Nano Sciences and Nano(bio)technology require: